Escherichia coli (E. coli) remains a major cause of economic losses for the pig industry. Therapy to combat PWD typically consists of antibiotic treatment or supplementation of zinc oxide to the feed. The emergence of antimicrobial resistance and new EU regulations prompt the need for alternative control strategies, such as immunization. The aim of the field study was to evaluate the effect of an oral live non-pathogenic E. coli vaccine on piglet performance, health, and antimicrobial use. We compared 10 batches receiving a standard antimicrobial control treatment to 10 batches vaccinated with the oral E. coli vaccine. The vaccine-treated groups demonstrated a significant improvement in performance, mortality weight, and antimicrobial use. In addition, secondary infections due to Streptococcus suis in the second part of nursery were reduced, as indicated by the reduction in amoxicillin use. In conclusion, the present study demonstrates the efficacy of an oral live non-pathogenic E. coli vaccine for the active immunization of piglets against PWD under field conditions. Therefore, vaccination against PWD may be considered a valuable alternative for strengthening piglet performance while meeting the new EU requirements concerning the prudent use of antimicrobials in intensive pig production.Post-weaning diarrhea (PWD) due to Escherichia coli (E. coli) remains a major cause of economic losses for the pig industry. Therapy to combat PWD typically consists of antibiotic treatment or supplementation of zinc oxide to the feed. The emergence of antimicrobial resistance to E. coli and new EU regulations prompt the need for alternative control strategies, such as immunization. The aim of the field study was to evaluate the effect of an oral live non-pathogenic E. coli vaccine on piglet performance, health, and antimicrobial use. We evaluated vaccination with an oral live non-pathogenic E. coli F4/F18 under field conditions in 10 consecutive batches against a standard antimicrobial treatment in 10 historical control batches. The vaccine-treated groups demonstrated a significant improvement in feed conversion rate, mortality weight, and antimicrobial use. From a general health perspective, secondary infections due to Streptococcus suis (S. suis) in the second part of nursery were markedly reduced, as indicated by the reduction in amoxicillin use. In conclusion, the present study demonstrates the efficacy of an oral live non-pathogenic E. coli vaccine for active immunization of piglets against PWD under field conditions. The vaccine-treated groups showed an improvement in several economically important performance parameters while reducing the overall antimicrobial use and infection pressure due to S. suis. Therefore, vaccination against PWD may be considered a valuable alternative for consolidating piglet performance while meeting the new EU requirements concerning the prudent use of antimicrobials in intensive pig production.Post-weaning diarrhea (PWD) due to  Escherichia coli (E. coli) (ETEC) has been identified as the most important cause of PWD. Two specific virulence factors typically characterize the ETEC pathotype, namely the presence of fimbrial adhesins, which mediate the attachment of bacteria to porcine intestinal enterocytes, and enterotoxins, which disrupt fluid homeostasis in the small intestine. Combined with a disruption in both the structure and absorptive function of the large intestine due to weaning-related stress [E. coli isolates from PWD [E. coli strains isolated from PWD over the last decades spurs the need for alternative control strategies [Post-weaning diarrhea (PWD) in pigs is recognized as an economically important disease worldwide . The mosd stress ,10, the d stress . The adhd stress . Other ffrom PWD ,14,15,16from PWD ,14,15,16rategies ,19,20,21E. coli in post-weaned piglets have been explored [E. coli during passage through the stomach [E. coli in particular [Several alternative strategies to optimize intestinal health and decrease the incidence of PWD due to explored ,23,24. Iexplored ,26,27. Aexplored . Other fexplored . Coarsel stomach . Other f stomach . The add stomach . Furtherrticular . Several decades ago, in the early 1980s, multiple studies on the supply of zinc to post-weaned piglets were performed. The supplementation of dietary zinc oxide (ZnO) has been proven to play an important role in the prevention and healing of PWD in several nutritional studies . Based oE. coli vaccination against PWD due to F4- and F18-ETEC, the prerequisite is that active mucosal immunity against both F4 and F18 is mounted prior to exposure to the pathogenic F4- and F18-ETEC strains. The activated mucosal immunity results in local production of F4- and/or F18-specific secretory IgA antibodies, which prevent pathogenic F4- and F18-ETEC from attaching to the intestinal F4- and F18-receptors, thus reducing the appearance of clinical signs of PWD [E. coli F4 or E. coli F4 and F18 vaccine has demonstrated efficacy against PWD due to F4-ETEC and F4- and F18-ETEC [E. coli F4 vaccine had increased weight gain [E. coli F4/F18 vaccine [Therefore, other preventive strategies toward PWD due to ETEC have recently been explored ,35. For F18-ETEC ,38,39,40F18-ETEC ,37. Moreght gain . A recenS. suis is one of the most important bacterial swine pathogens affecting post-weaned piglets. Streptococcal disease in this animal category is mainly characterized by meningitis, arthritis, and sudden death [E. coli, Actinobacillus pleuropneumoniae) pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field [S. suis is high, the incidence of disease can vary from period to period and is usually lower than 5%, mainly due to prophylactic or metaphylactic measures. This approach is in contrast with the current trend to reduce antimicrobial use in animals and humans as much as possible to prevent further development of antimicrobial resistance [S. suis infection are poorly understood, many virulence factors have already been identified that play a role in the crucial steps of infection development [S. suis bacteria spreading into the blood stream [Besides PWD due to F4- and F18-ETEC, en death . Co-infehe field . Even whsistance . Althougelopment . Followid stream . There, E. coli F4/F18 vaccine  for active immunization of piglets against PWD caused by F4-ETEC in 10 consecutive batches against a control group treated with the standard therapeutic approach using antimicrobial therapy. We evaluated piglet performance, piglet health related to secondary infections due to Streptococcus suis, and overall antimicrobial treatment during the post-weaning period.Here, we report results demonstrating the efficacy of an oral live non-pathogenic E. coli–C. perfringens–C. novyi vaccine . Gilts were vaccinated twice at 6 and 3 weeks prior to farrowing with the same commercial vaccine. Piglets were weaned at 22 days of age and housed in specifically equipped post-weaning facilities, where they were raised for 6.5 to 7 weeks (46 to 50 days post-weaning). The post-weaning facility was divided into five compartments of 28 pens each, which could house 20 post-weaned piglets. Dry feeders with two waterers, one on each side, were in the pen division, thus feeding two pens with a total of 40 piglets. The pens were further equipped with fully slatted plastic floors and were heated with hot water tubes located in the ventilation duct. Ventilation was performed through one ventilation fan evacuating the air into a central ventilation duct, and fresh air entered each compartment indirectly through the central corridor after being pre-heated if necessary.The field trial was performed on a conventional farrow-to-finish pig farm with 1000 TN70 sows in Flanders (Belgium). The farm was managed in a 4-week batch management system with 160 sows per production batch. This management approach has been shown to improve the health status for several respiratory pathogens . Sows wen = 10) with typical clinical signs of PWD, such as watery diarrhea, a thin belly, and signs of dehydration, were sampled using rectal swabs . All sampled piglets were between 3 and 5 days post-weaning. The diagnostic samples were sent to the laboratory  under cooled conditions for further processing. The farm suffered for several years from PWD outbreaks due to ETEC in each consecutive batch. The farm was selected following ETEC diagnostics during the post-weaning period. Therefore, untreated piglets , F5 (K99), F6 (987P), F18, F41, LT, STa, STb, and Stx2e, but not discriminating between F4ab, F4ac, and F4ad. The methodology used for identification of these virulence genes has been described previously [DNA samples were prepared from one up to five hemolytic and/or non-hemolytic eviously . All colS. suis with a resistance to several antimicrobials applied during treatment of streptococcal meningitis, such as tetracycline, doxycycline, and lincomycin  for necropsy and further analysis. All cases were confirmed positive for ncomycin . E. coli F4/F18 vaccine  has a rapid onset of immunity (OOI) of only 7 days and a duration of immunity (DOI) of 21 days post-vaccination, which covers the most important critical period for the occurrence of PWD [E. coli F4 challenge at 3 days post-vaccination demonstrated a reduction in both the severity and duration of PWD and a reduction in fecal shedding of pathogenic F4-ETEC [E. coli F4/F18 vaccine during the 7 days following vaccination. To vaccinate piglets at least 7 days prior to the clinical signs, in order to mount sufficient protective local immunity in the gut , piglets F4-ETEC . The vacE. coli F4/F18 vaccine reduced the excretion of pathogenic E. coli bacteria in the vaccine-treated group [E. coli F4/F18 vaccine, as described previously. The field trial was carried out between 16 November 2020, when the first control group was enrolled, and 20 June 2022, when the last vaccine-treated group was finalized. Due to the practical setting, it was not possible to run control and vaccine-treated groups concurrently in one batch of weaned piglets. Moreover, since vaccination with the ed group , alternan), number of piglets moved to the next phase (n), date of weaning and end of nursery period, piglet body weight  at d0 and at the end of the nursery period (d46-50) (kg/piglet), feed intake during the nursery period (kg/piglet), and mortality  with weight at death (kg).The following performance parameters were collected during the trial: number of piglets enrolled in each batch . Based on these parameters, the following performance and health indicators were calculated: number of days in nursery (d), average daily weight gain , average daily feed intake , feed conversion rate , percentage of piglets transferrable to the next phase (%), treatment cost (€/piglet), and treatment incidence over a 100-day period in the nursery  was applied. For the continuous data, the effect of the treatment on the different outcome parameters was assessed using a t-test with pooled standard deviations. All tests were performed at the nominal level of 5%.p = 0.386) different between the control group  and the vaccine-treated group . The total sold piglet weight was not significantly (p = 0.414) different between the control group  and the vaccine-treated group . The average percentage of sold piglets per batch was numerically, but non-significantly (p = 0.094), higher in the vaccine-treated group (97.1 ± 0.2%) than the control group (96.5 ± 0.4%) (p = 0.406) different between the control group (6.61 ± 0.22 kg) and the vaccine-treated group (6.53 ± 0.23 kg). The average BW at selling was not significantly (p = 0.063) different between the control group (23.97 ± 0.41 kg) and the vaccine-treated group (23.18 ± 0.26 kg) (p = 0.027) shorter in the control group (46.7 ± 0.5 d) than the vaccine-treated group (49.6 ± 0.6 d). The total weight gain (WG) from weaning to selling was similar in both treatment groups  (p = 0.129). Nevertheless, the ADWG was significantly (p = 0.008) higher in the control group (375 ± 12 g) than the vaccine-treated group (336 ± 6 g)  . The ave0.26 kg) . The per6 ± 6 g) .p = 0.052) different, in the control group (28.98 ± 1.00 kg) than the vaccine-treated group (26.84 ± 0.56 kg). The ADFI was significantly (p = 0.008) lower in the vaccine-treated group (540 ± 9 g/d) than the control group (625 ± 27 g/d) (p = 0.041) better in the vaccine-treated group (1.61 ± 0.02) than the control group (1.67 ± 0.02)  per sold piglet  was higher, but not significantly ( 27 g/d) . The FCR ± 0.02) .p = 0.181). At selling, an average of 2541 (±68) and 2589 (±68) piglets per batch were eligible to move to the next phase in the control and vaccine-treated groups, respectively (p = 0.157)  and 2720 (±98) piglets were weaned per batch in the control and vaccine-treated group, respectively (= 0.157) .p = 0.133). In the control group, 3.52 (±0.38) % of the piglets died during the nursery period, whereas in the vaccine-treated group 2.90 (±0.25) % died during the same period. The mortality rate (expressed as a percentage) was not significantly different between both groups (p = 0.094)  and 79 (±6) piglets died per batch during the entire nursery period in the control and vaccine-treated groups, respectively. This number was not significantly different between both treatment groups (= 0.094) . Neverthp = 0.00013) lighter in the vaccine-treated group (5.46 ± 0.42 kg) than the control group (7.80 ± 0.26 kg) (The average weight of dead piglets was significantly (0.26 kg) . 100, or the number of days on treatment for every 100 days in nursery, was significantly (p = 0.000022) higher in the control group (69.42 ± 9.44 d) than the vaccine-treated group (0.13 ± 0.13 d)  . This res result . From Fi batches .p = 0.0086) was observed between the control group (€ 0.36 ± 0.11) and the vaccine-treated group (€ 0.05 ± 0.02). This represents an 86.1% reduction in the antimicrobial treatment costs between the control and vaccine-treated group  reduction in the weight of dead piglets between the control group (7.80 ± 0.26 kg) and the vaccine-treated group (5.46 ± 0.42 kg) and by the 91.5% reduction in the amount of amoxicillin used for the treatment of streptococcal meningitis in the second phase of the nursery period between the control group (59 kg over 10 batches) and the vaccine-treated group (5 kg only used in the first vaccinated batch)  . In addiy period . 100, were significantly improved in the vaccine-treated group as compared to the control group. The current field study on the vaccination of piglets pre-weaning to protect against PWD due to F18-ETEC clearly demonstrates that the overall technical performance was not significantly different between both treatment groups, considering that the control group had a significantly higher antimicrobial use than the vaccine-treated group. Several economically important performance parameters, such as ADFI, FCR, antimicrobial treatment cost, and TI100 from 69.43 d to 0.13 d. It has been shown that even therapeutic use of antimicrobials results in an improvement in animal growth efficiency through inhibition of the normal gut microbiota, eventually leading to increased nutrient utilization and reduction in the maintenance cost of the gastro-intestinal system [The total WG in nursery was only 600 g lower in the vaccine-treated group over the entire nursery period of about 50 days. Several factors might explain this slight, but non-significant, difference. In the vaccine-treated groups, about 90 supplementary piglets were weaned per batch, which may explain the lighter weaning weight of 90 g per piglets in this group. Another important factor that may have contributed to the slightly lower total WG in the nursery period was the significant reduction in antimicrobial use observed both for the treatment and control of PWD in the first phase of nursery and streptococcal meningitis in the second phase of nursery , which rl system .For FCR, an improvement of 0.06 in piglet performance represents an approximate economic advantage of about 3.7 eurocent per kg gain—at the current average market value of EUR 620 per ton of post-weaning piglet feed—or 1.05 kg less feed per piglet for the same post-weaning WG. Feed cost should be considered the most important economic aspect in piglet production; the above calculated benefit may already have a significant impact on the net farm’s income during the post-weaning production phase. In fact, the significantly lower ADWG (−39 g/d) in the vaccine-treated group is therefore largely compensated for by a significantly lower ADFI (−85 g/d), which could result in this significant improvement in the FCR in the vaccinated piglets. These results are in accordance with a recent analysis of 10 field trials in Belgium and the Netherlands in which a similar significant improvement in FCR could be observed . In contE. coli vaccine is 7 days, and piglets of different litters are commingled according to their weight and general condition at weaning. Changes in these day-to-day routine management aspects would complicate the set-up and performance of this practical field trial and might lead to involuntary and non-detectable errors that may have blurred the results and conclusions. Use of the current study design allowed us to change only one specific parameter—i.e., vaccination of piglets prior to weaning—and evaluate the effects of this implemented strategy with changes to any other management practices. The current field study was carried out using a historical control group, which could be considered a minor negative aspect. However, from a practical point of view, the current farm did not allow us to run concurrent treatment groups at the same time due to several practical constraints. Although several compartments were available in the nursery phase, only one automated feed line was available to distribute the feed to all compartments. In addition, the construction of the barn did not efficiently allow for separation of both treatment groups with a sufficient level in internal biosecurity to omit the spread of pathogens between both treatment groups. A third practical issue was that the piglets had to be vaccinated pre-weaning, since the onset of immunity of the oral live non-pathogenic www.abregister.be (accessed on 15 July 2022)) by the farm veterinarian on a quarterly basis. Based on these registrations and the individual delivery documents of the antimicrobial products, we were able to analyze the TI100 and details of all administered products for both clinical conditions  at the farm. The current study demonstrated a drastic decrease in antimicrobial use following implementation of an oral live non-pathogenic E. coli vaccine in piglets to prevent the clinical signs of PWD due to F18-ETEC. Indeed, both the overall TI100 and the more detailed data per batch showed a marked decrease in antimicrobial use following vaccination. The average amount of antimicrobial prescribed and used for the treatment of PWD decreased from 5.6 kg per batch to 0.0 kg, which resulted in nine consecutive batches with a TI100 of zero. The only antimicrobial used following vaccination was amoxicillin in the first vaccinated batch for the treatment and control of streptococcal meningitis due to S. suis. However, treatment of this clinical condition was not needed from the second vaccinated batch onward. These results are in accordance with previous studies using the E. coli vaccine [100—total number of treatment days per 100 days in nursery—can be registered. In Belgium, antimicrobial use at the farm and animal category level  is registered in a central database  was observed in the vaccine-treated group as compared to the control group. Of the total number of weaned piglets , this reduction in mortality resulted in an increase in the number of piglets that could move to the next phase , which may also be considered an important economic benefit following vaccine implementation. S. suis. The decrease in the average BW of dead piglets was mainly due to the decrease in second phase mortality related to these clinical problems with streptococcal meningitis, which can be confirmed by the antimicrobial consumption data of amoxicillin in the vaccine-treated group. Only in the first vaccinated batch was amoxicillin needed for the treatment and control of streptococcal meningitis due to S. suis. Thereafter, no amoxicillin was prescribed or used in the vaccine-treated group. A potential explanation may be found in the improved intestinal integrity following an oral live non-pathogenic E. coli vaccination, which may result in a lowered opportunity for S. suis to colonize the host and subsequently progress through the intestinal mucous membranes using its suilysin to finally spread through the blood to the meninges after passing the blood–brain barrier [S. suis as a secondary pathogen following initial viral or bacterial infections have been reported [S. suis. Moreover, induction of this specific combination of co-infection between PWD due to ETEC and streptococcal meningitis due S. suis is difficult to reproduce under experimental conditions. Another remarkable aspect of the mortality data, besides the slightly lower mortality in the vaccine-treated group, is that the average BW of dead piglets is significantly lower (−2.36 kg per dead piglet) in the vaccine-treated group than in the control group. In practice, it is known that light-weight piglets predominantly die in the first 2 weeks post-weaning from PWD due to F18-ETEC and other problems related to livability due to their low weaning weight, whereas heavier piglets predominantly die later during the nursery period due to streptococcal meningitis due to  barrier . Althougreported , little E. coli vaccine for the active immunization of piglets against PWD due to F18-ETEC under field conditions. Vaccine-treated groups showed improvements in several economically important performance parameters while reducing the overall antimicrobial use and infection pressure due to S. suis. Therefore, vaccination against PWD may be considered a valuable alternative for strengthening piglet performance while meeting the new EU requirements concerning the prudent use of antimicrobials in intensive pig production.The current field study reports the efficacy of an oral live non-pathogenic


The elasticity of RBOB-FG emulsion-filled gels and the ball-bearing effect led to a minimum boundary friction coefficient (μ). These results might contribute to the development of oil-body-based functional ingredients for applications in plant-based foods as fat replacements and delivery systems.In this study, rice bran oil bodies (RBOBs) were filled with varying concentrations of flaxseed gum (FG) to construct an RBOB-FG emulsion-filled gel system. The particle size distribution, zeta potential, physical stability, and microstructure were measured and observed. The molecular interaction of RBOBs and FG was studied by Fourier transform infrared spectroscopy (FTIR). In addition, the rheological and the tribology properties of the RBOB-FG emulsion-filled gels were evaluated. We found that the dispersibility and stability of the RBOB droplets was improved by FG hydrogel, and the electrostatic repulsion of the system was enhanced. FTIR analysis indicated that the hydrogen bonds and intermolecular forces were the major driving forces in the formation of RBOB-FG emulsion-filled gel. An emulsion-filled gel-like structure was formed, which further improved the rheological properties, with increased firmness, storage modulus values, and viscoelasticity, forming thermally stable networks. In the tribological test, with increased FG concentration, the friction coefficient ( Oil bodies (OBs), also known as oleosomes, are micron- or submicron-sized natural oil droplets . OBs areRice bran oil bodies (RBOBs) are natural pre-emulsified emulsions composed of a triacylglycerol core surrounded by a phosphate layer embedded with various kinds of oleosins and some minor proteins of higher molecular mass . ExtractEmulsion gel contains both emulsion droplets and gels . EmulsioIn this study, RBOBs in rice bran were extracted by the aqueous method. Two types of RBOB-FG emulsion-filled gels system (fluid emulsion gels/bulk emulsion gels) were constructed using three concentrations of FG . The particle size distribution, zeta potential, physical stability, and microstructure of the RBOB-FG emulsion-filled gels were measured and observed. The molecular interaction of RBOBs was studied by Fourier infrared spectroscopy. The rheological properties and the tribology of the RBOB-FG emulsion-filled gels were also evaluated. This research may contribute to the development of a new and simple strategy to determine the structure of RBOBs, which can be used in the development of fat replacements and edible soft solid materials.Rice bran was purchased from Shuyang Runyi Agricultural Technology Co., Ltd. . FG was obtained from Yuanye Biological Company  and used without further treatment. The FG contained 70.92% polysaccharide, 12.67% protein, and 16.31% ash, as supplied by the manufacture. Nile blue A and Nile red were provided by Sigma-Aldrich Co. . All other chemicals were of analytical grade. All solutions and emulsions were prepared using ultrapure water .Coarse OBs were physically isolated from a homogenate of rice bran according to the method described in , with a An RBOB emulsion was prepared by dispersing extracted oil body creams into phosphate-buffered solution  under magnetic stirring. Varying contents of FG were mixed into RBOB emulsion, heated at 50 °C in a water bath, and magnetically stirred for 1.5 h. The final oil concentration was 5.0 wt.%, and the concentration of FG was 0.2, 0.8, and 2.0 wt.% for each of the mixtures, respectively. The pH of the RBOB-FG emulsion-filled gels was determined to be 6.5. Then, all samples were stored in refrigerator  for gelation to obtain RBOB-FG emulsion-filled gel. For comparison, FG hydrogel without RBOBs was prepared as a control at pH 6.5, using NaOH (1.0 M) and HCl (1.0 M). Concentrations of 0.2, 0.8, and 2.0% FG were used for the gel matrix because we expected to obtain liquid and solid-like gel samples for food applications.For analysis of particle size and ζ-potential, average particle size and distribution were measured using SDC-Microtrac S3500 laser diffraction equipment  with 3 readings for each sample. Zeta potentials of the droplets were measured with a Nano-ZS90 zetasizer  through the assessment of droplet velocity and the direction in the electrical field. Phosphate-buffered solution (10.0 mmol/L) was used to dilute the measured samples with the same pH as the samples to avoid multiple scattering effects. All samples were equilibrated for 120 s before obtaining data.A LUMiSizer  was used to measure the physical stabilities of RBOB-FG emulsion-filled gel, RBOB emulsion, and FG hydrogel. After storage at 4 °C storage for 12 h, samples were accelerated to test their instability  according to the method described in .−1 in absorption mode, with 32 scans and a resolution of 4 cm−1.A Bruker Tensor II instrument  was used to acquire infrared spectra of RBOB-FG emulsion-filled gel, FG hydrogel, and RBOBs at 25 °C. PBS was used as the blank. All samples were freeze-dried with KBr and pressed into thin sheets. Samples were tested in the scanning range of 500–4000 cmThe microstructure of dried and structured RBOB-FG emulsion-filled gel samples were analyzed using scanning electron microscopy . The accelerating voltage was 1.2 kV during the measurements. A small portion of sample was placed on an aluminum holder using a double-adhesive conductive pad. Loose particles were removed by spraying dry air on the stent surface. RBOBs, RBOB-FG emulsion-filled gel, and FG hydrogel were observed with an IX81 light microscope .A CLSM  was used to observe the microstructure of samples. The TAG cores of RBOBs were stained with Nile red, and RBOB proteins were stained with Nile blue A . All samThe microrheological properties of samples were measured by Rheolaser Master  based on the diffusing wave spectroscopy theory. A 3 h microrheology test was carried out on RBOBs, RBOB-FG emulsion-filled gel, and FG hydrogel.−1. The experimental curve was fitted using the Herschel–Bulkley model represented by −1), K is the consistency index (Pa sn), and n is the flow behavior index.After the gelation , the rheological properties of the FG hydrogel/RBOB-FG emulsion-filled gels were characterized at 25 °C on a Haake 6000 RheoStress rheometer . A 35 mm diameter parallel plate with waterproof sandpaper was used for measurement . The visBefore starting the experiment, low-viscosity silicone oil was used to prevent the water from evaporating. Samples were loaded onto the rheometer, which was preheated to 90 °C. After an equilibration time of 5 min, a temperature sweep was processed. A cooling step from 90 to 20 °C and a heating step from 20 °C to 90 °C were performed at a constant rate of 1 °C/min. The process was examined by monitoring the G′ (storage modulus) and G″ (loss modulus) change under a fixed frequency (1 Hz) and 1.0% strain.A TA-DHR friction rheometer was used with a full-ring stainless steel probe, and the surface of the oral cavity was simulated with polydimethylsiloxane (PDMS) to measure the lubricity of the particles. The rotating sphere automatically adjusted with a normal force (3 N) evenly distributed on the lower plate at 37 °C. As the sliding speed increased from 0.1 to 450 mm/s, the friction force between the stainless ball and the plates was recorded. The coefficient of friction was derived using the following formula: coefficient of friction = friction force/set normal force.w/w) to prepare PDMS (Sylgard184) with a surface roughness (Ra) < 50 nm, vacuum to remove bubbles generated during mixing, and smooth container stainless steel mold; cure overnight at 70 °C. A rotational rheometer mounted with an accessory based on 3-ball plate tribology geometry was used for tribological tests [PDMS (Sylgard184) production method: mix base fluid and cross-linking agent .The average values and the standard deviation (SD) were reported using descriptive statistical analysis, which was performed through SPSS statistics, version 25 .The particle size distributions of RBOB-FG emulsion-filled gel formed by RBOB emulsions mixed with varying FG concentrations  at pH 6.5 are shown in The ζ-potentials of RBOB-FG emulsion-filled gels are displayed in −1 and C-H stretching at 3200–2800 cm−1, as well as peaks characteristic of polysaccharides at 1644–1630 cm−1 and 1036 cm−1. The characteristic absorption peaks near 849 and 831 cm−1 in the spectrum indicated that there were glycosidic bonds [−1 and 1738 cm−1 , 1454 cm−1(C-H deformation), and 1200–1100 cm−1(C–O bonding) [−1, 2851 cm−1, and 1744 cm−1. As portrayed in −1(C=O). A new peak appeared at 1711 cm−1, with the amide I and II bands of RBOB proteins shifted from 1644.07 to 1650.56 cm−1 and from 1548.34 to 1544.53 cm−1, possibly due to the hydrogen bonding interaction between RBOB emulsion and FG [The molecular interaction between RBOB emulsion and FG was studied by FTIR . FG exhiic bonds . With ren and FG . The aboThe microstructures of RBOB emulsion, FG hydrogel, and RBOB-FG emulsion-filled gel microparticles were investigated by SEM, as shown in CLSM micrographs of RBOB-FG emulsion-filled gels/FG hydrogels with varying FG concentrations and neutral pH are shown in dec). The elasticity of the sample with shorter decorrelation time (0 < tdec < 1.5 s) was represented by the height of the MSD curves. With a short decorrelation time, a downward movement of RBOB-FG emulsion-filled gel and FG hydrogel MSD curves was observed, indicating that collision between particles, viscoelasticity, and interactions in the gel network were gradually enhanced in the system.The microrheological properties of RBOB-FG emulsion-filled gels/FG hydrogels with varying FG concentrations were investigated . In the The MSD curves show that a stronger elastic system of RBOB-FG emulsion-filled gel was formed. Compared to FG hydrogels, the droplet movements were limited within RBOB-FG emulsion-filled gel due to the high viscosity.To study the effect of RBOB emulsion on the rheological properties of varying FG concentrations, a frequency sweep and temperature sweep were performed on the RBOB-FG emulsion-filled gels and FG hydrogels. Storage modulus (G′) and loss modulus (G″) are commonly used indicators to characterize the gel properties . G′ refe−1, reflecting progressive shear-induced breakdown of structure in RBOB-FG emulsion-filled gel , as well as the G′ and G″ values of the RBOB-FG emulsion-filled gel and the FG hydrogels, were determined. Compared to FG hydrogel  was not observed with 2.0 wt.% FG content in RBOB-FG emulsion-filled gels (m) of RBOB-FG emulsion-filled gels  and sliding speed (S) for FG gel  and pure water is shown in μ plateau up to S = 60 mm/s and a maximum value of friction coefficient (μ) of between 1.42 and 1.93. Since PDMS films are hydrophobic, so water is unable to form a continuous lubricating interface. With a boundary lubrication regime up to 3.8 mm/s, μ values of 0.2%, 0.8%, and 2.0% FG hydrogels were 0.82–0.86, 0.42–0.47, and 0.30–0.37, respectively, suggesting that the fragments generated by the FG hydrogel were entrained into the contact surface during frictional sliding, reducing surface roughness and thus improving the lubrication.The functional relationship between friction coefficient (S), the μ value of the FG hydrogels  decreased to around 0.024–0.045, and those of pure water decreased to about 0.02 at a rate of 450 mm/s. In the examined sliding speed range, neither pure water nor the FG hydrogel showed a hydrodynamic lubrication regime. During the mixed lubrication regime, a reduction in the μ value of the FG hydrogels was observed, possibly due to the FG breaking into more fragments with increased S values, resulting in large amounts of FG fragments being entrained to the friction interface [μ value in this regime than the FG gel due to the relatively higher G′ of the RBOB-FG emulsion-filled gel. Because the RBOB-FG emulsion-filled gel had a higher gel strength and a more stable network structure, it was not easily deformed when extruded by a perpendicular load between two contact surfaces, which increased rough surface separation and reduced friction. As shown in μ of the RBOBs increased from 0.04 at S = 150 mm/s to 0.088 at S = 150 mm/s during the boundary lubrication regime. According to the plate-out theory explained in [μ value. However, no obvious oil leakage was observed after the tribology test. Because the viscoelastic RBOB droplets easily entrained, filling between the two surfaces reduced the friction at a slow sliding speed.With increased sliding speed , and fragments of FG matrix containing RBOBs were entrained into the contact surfaces, reducing the μ value. First, the leaked RBOB droplets acted as small, spherical bearings that can scroll and bear loads [S > 90 mm/s, more fragments were entrained into the contact surfaces, and parts of the gel fragments may have squeezed and flipped over each other between the gaps, resulting in an increase in μ.The “bearing balls” theory considers RBOBs as pre-emulsion droplets with a similar spherical shape and small particle size . Therefoar loads . Then, tA schematic diagram is presented to illustrate the formation of RBOB-FG emulsion-filled gels . Due to RBOB-FG emulsion-filled gel was produced with FG as the gel matrix and filled with RBOB droplets. The result show that negatively charged FG enhanced electrostatic repulsion, and steric hindrance facilitated the physical stability of the droplets, changing the structure of RBOB protein through the formation of hydrogen bonds. The microstructure of RBOB droplets in the FG hydrogel gel matrix showed good distribution and formed an RBOB-FG emulsion-filled gel structure. RBOBs improved the storage modulus, viscoelasticity, melting, and solidification temperature, indicating that RBOBs can function as an active filler, strengthening networks. The ball-bearing effect of RBOBs and higher storage moduli values of RBOB-FG emulsion-filled gel are believed to contribute to improved lubricating properties relative to FG gels. The present work provides insights on the behaviors of RBOB emulsion-filled gel, which can be applied to design strategies for practical processes to obtain OB emulsion gels in food structuring and as fat replacements in fresh sausages, salad dressings, and bakery products.


Exosome–liposome hybrid nanocarriers containing chemotherapeutic agents have been developed to enhance drug delivery, improve the efficacy of the treatment of metastatic cancer, and overcome chemoresistance in cancer therapy. Thus, the objectives of this study were to investigate the toxicological profiles of exosomes fused with long-circulating and pH-sensitive liposomes containing doxorubicin (ExoSpHL-DOX) in healthy mice and the antitumor activity of ExoSpHL-DOX in Balb/c female mice bearing 4T1 breast tumors. The acute toxicity was determined by evaluating the mortality and morbidity of the animals and conducting hematological, biochemical, and histopathological analyses after a single intravenous administration of ExoSpHL-DOX. The results of the study indicated that the ExoSpHL-DOX treatment is less toxic than the free doxorubicin (DOX) treatment. ExoSpHL-DOX showed no signs of nephrotoxicity, even at the highest dose of DOX, indicating that the hybrid nanosystem may alter the distribution of DOX and reduce the kidney damage. Regarding the antitumor activity, ExoSpHL-DOX showed an antitumor effect compared to the control group. Furthermore, the hybrid nanocarrier of tumor-derived exosomes fused with long-circulating and pH-sensitive liposomes reduced the number of metastatic foci in the lungs. These results indicate that ExoSpHL-DOX may be a promising nanocarrier for the treatment of breast cancer, reducing toxicity and inhibiting metastasis, mainly in the lungs. Cancer is one of the leading causes of death and an important barrier to efforts to increase life expectancy across the world. Annually, more than 19 million people develop cancer and approximately 10 million people die from the disease. Breast cancer is the most commonly diagnosed cancer in women, with 2.3 million new cases . However2000) were supplied by Lipoid GmbH . Cholesterol hemisuccinate (CHEMS), DOX, phosphate-buffered saline (PBS), sodium hydroxide, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and sodium bicarbonate were obtained from Sigma-Aldrich . Total exosome isolation reagent was obtained from Thermo Fisher Scientific .1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEGThe 4T1 murine breast cancer cells were purchased from the American Type Culture Collection (ATCC) . Roswell Park Memorial Institute (RPMI) 1640 Medium and fetal bovine serum (FBS) were obtained from Gibco Life Technologies . Trypsin was obtained from Sigma-Aldrich . A mycoplasma test using Hoechst fluorescence staining was performed on the cell line.2 in a humidified atmosphere. When the 4T1 cells reached an approximate confluence of 80%, the supernatant was removed from the cell culture flask T-75. The exosome isolation reagent was added to the supernatant  and kept in a refrigerator for 15 h. After that time, the mixture was centrifuged at 10,000× g for 1 h at 4 °C using a centrifuge from Thermo Scientific, model Heraeus Multifuge X 1R. The pellet was dissolved in a mixture of chloroform and methanol (1:1 v/v ratio).The 4T1 cells were grown in RPMI-1640 supplemented with 10% of ultracentrifuged FBS and maintained at 37 °C and 5% CO2000  and exosomes in a mixture of chloroform and methanol were added to a round-bottomed flask to obtain a lipid film. For each mL of liposome, we added an exosome pellet obtained from 2 mL of cell supernatant (concentration of 3.6 × 1010 particles/mL). After the evaporation of the solvents, NaOH 0.228 M solution was added to ionize the CHEMS molecules and, subsequently, promote the formation of vesicles. The hydration of the lipid film was carried out under agitation with an ammonium sulfate solution . The vesicles obtained were calibrated by extrusion using the Lipex Biomembranes extruder, Model T001  [® L-80XP, Beckman Coulter, Brea, CA, USA) at 150,000× g, 4 °C, for 120 min. The pellet was resuspended with HEPES buffered saline (HBS). The vesicles were incubated with a DOX solution for 2 h in the dark at room temperature. The non-encapsulated DOX was removed by ultracentrifugation using the same method as that described above. The final pellet was resuspended with HBS. Blank breast-tumor-derived exosomes fused with long-circulating and pH-sensitive liposomes (ExoSpHL) and long-circulating and pH-sensitive liposomes containing DOX (SpHL-DOX) were prepared in the same way without the addition of DOX and exosomes, respectively.ExoSpHL-DOX was prepared using the Bangham method  followed Canada) . The extThe mean diameter and the polydispersity index (PDI) of ExoSpHL-DOX were measured by dynamic light scattering (DLS). The zeta potential value was determined by DLS combined with electrophoretic mobility. To perform both analyses, 50 µL of ExoSpHL-DOX was diluted in 1 mL of HBS, and the Zetasizer Nano ZS90 equipment was used .v/v). Samples were injected (20 μL), and the separation was performed with an ACE® C8 column, 25 cm × 4.6 mm, 5 μm , at a flow rate of 1.0 mL/min. The detection was performed in the model 2475 fluorescence mode , with excitation and emission wavelengths of 470 nm and 555 nm, respectively [v/v, respectively) and diluted in the mobile phase. The encapsulation percentage (EP) of DOX in ExoSpHL-DOX was calculated according to the following equation:The DOX content was measured by high-performance liquid chromatography (HPLC). The mobile phase consisted of methanol:phosphate buffer pH 3.0 . The mice were kept in plastic cages with free access to food and water and under standardized light/dark cycle conditions. All protocols were approved by the Ethics Committee for Animal Experiments from the Universidade Federal de Minas Gerais (CEUA/UFMG—protocol number 265/2019).The acute toxicity was assessed according to the recommendations of the Organization for Economic Cooperation and Development (OECD) 423 , adaptedFor the hematological analysis, the blood was collected in tubes containing anticoagulant (EDTA 0.1 M) and inserted into the automated hematological analyzer HEMOVET 2300 . Hematological parameters related to red and white blood cells were evaluated for each treatment group. For the biochemical analysis, the blood was centrifuged , and the plasma obtained was collected. The tests were performed with the Bioplus BIO-2000 semiautomatic analyzer  using commercial kits . The renal, liver, and cardiac functions were evaluated for each treatment group.w/v in phosphate-buffered saline (PBS), pH 7.4) and incorporated in paraffin blocks. Consecutive histological sections were prepared and stained by the hematoxylin and eosin routine method. The slides were evaluated by trained pathologists, and images of histological sections were captured using a digital camera connected to an optical microscope, Olympus BX-40 .The liver, kidneys, spleen, and heart were harvested and fixed in formalin (10% 6 cells in 100 µL PBS). When the tumor volume reached approximately 100 mm3, the animals were randomly divided into five treatment groups, each containing six animals. Each group intravenously received five administrations of HBS, ExoSpHL, free DOX, SpHL-DOX, or ExoSpHL-DOX. The cumulative dose of DOX was 25 mg/kg. The dose used in this study was based on a previous study of our research group [2), d1 and d2 being the largest and the smallest perpendicular diameters, respectively. Six measurements of the diameters of the tumors were carried out during ten days of treatment using a caliper MIP/E-103 . The TV at day 0 was considered as 100%, and changes in the TV were determined every two days by calculating the percentages of the TV increase or decrease. The relative tumor volume (RTV) and inhibition percentage of tumor growth (TGI) were calculated according to the following equations:The 4T1 breast cancer cells were injected into the right flank of the female Balb/c mice  and ketamine (80 mg/kg). The liver, spleen, lungs, heart, and tumor were collected for the histopathological analysis.p < 0.05 were considered significant. The analyses were performed using the GraphPad Prism software .To confirm the normality and homoscedasticity of variance, D’Agostino and Shapiro–Wilk tests were applied, respectively. The differences between the experimental groups were tested by analysis of variance (one-way ANOVA followed by Tukey’s test). If the data were not normal or homoscedastic, the Kruskal–Wallis test with Dunn’s post-test was used for the same purpose. The two-way ANOVA test with Tukey’s post-test was also used to relate two different independent variables with respect to one dependent variable. Values of ExoSpHL-DOX presented a mean diameter of 105.4 ± 2.9 nm and a PDI value of 0.132 ± 0.010, indicating the presence of monodisperse vesicles. The zeta potential value was near neutrality (−6.4 ± 1.2 mV), as expected for vesicles that contain PEG in their composition. The encapsulation percentage of DOX was 88.5 ± 2.4%, achieved by using the ammonium sulfate gradient method.The HBS and ExoSpHL treatments did not show significant differences in relation to the mortality and morbidity. These findings indicate the lack of toxicity of the treatments applied to the mice in the control groups. The LD50 assessment of animals treated with free DOX started with 10 mg/kg and with 15 mg/kg for both formulations (SpHL-DOX and ExoSpHL-DOX). For the free DOX treatment, the 10 mg/kg dose showed no significant signs of toxicity in the first three animals tested. Thus, the next step was the dosing of three additional animals with the same dose. The results remained the same in all animals; therefore, the following higher dose level was injected into three animals. After 8 days of application of 12.5 mg/kg of DOX, piloerection and ascites in the animals were observed. It is worth mentioning that the most notable result was a weight loss of 13%, observed at day 12 post-administration. However, no deaths were observed. Therefore, the next step was the dosing of three additional animals with the same dose. The previous observations were confirmed, and there was one death on day 13. According to The studies carried out using the SpHL-DOX and ExoSpHL-DOX treatments started with a dose of 15 mg/kg. For both treatments, there was no death or weight loss in all of the six animals tested. After treatment with a dose of 17.5 mg/kg, no deaths were observed. However, there was a 5% weight loss in the animals. According to these results, we repeated the experiments for the groups treated with SpHL-DOX and ExoSpHL-DOX at a dose of 17.5, using three more animals per treatment, and the previous observations were confirmed. We followed the treatment scheme , increasThe hematological parameters of the mice treated with DOX, SpHL-DOX, and ExoSpHL-DOX are shown in The biochemical parameters of the mice treated with DOX, SpHL-DOX, and ExoSpHL-DOX are shown in Histological analyses of the different organs were performed at the end of the treatment period. The mice treated with HBS and ExoSpHL presented with the same histopathological profile and, therefore, only the HBS treatment group’s photomicrography was presented as the control group. The liver analysis showed no changes in the SpHL-DOX (15 mg/kg) and ExoSpHL-DOX (15 mg/kg) treatment groups compared to the control group A. In conThe histopathological analysis of the kidneys revealed no changes in the case of the SpHL-DOX (15 mg/kg) and ExoSpHL-DOX (15 and 17.5 mg/kg) treatment groups compared to the control group A. AnimalThe cardiac muscle analysis revealed that, after the DOX treatments, areas of cardiomyocyte vacuolization were observable. Compared to the control group A, the trThe antitumor efficacy of the free DOX, SpHL-DOX, and ExoSpHL-DOX treatments was evaluated in the female Balb/c mice with 4T1 breast tumors by assessing the tumor volume variation over time. The tumor volume in the HBS (control) and ExoSpHL treatment groups increased rapidly over time and showed no significant differences between them. By contrast, significant differences in the tumor volume were observed between the control treatment groups and the groups treated with DOX, SpHL-DOX, and ExoSpHL-DOX at a dose of 5.0 mg/kg A. The tuThe body weight loss after the treatments with DOX, SpHL-DOX, and ExoSpHL-DOX was evaluated in the Balb/c female mice with 4T1 breast tumors. The results are presented in Histological analyses of the tumors and different organs were performed at the end of the treatment period. The 4T1 tumor cells grow in a solid arrangement. The proliferation of pleomorphic cells and a high mitotic index were observed . The micThe 4T1 murine breast cancer is highly tumorigenic and invasive, wherein metastatic foci are observed in various organs . The lunThe macroscopic analysis of the spleen showed splenomegaly in the animals treated with HBS or ExoSpHL, which is commonly observed in mice with 4T1 murine breast cancer. In the DOX, SpHL-DOX, and ExoSpHL-DOX treatments groups, the spleen size was normal, indicating that the treatments were able to reverse the splenomegaly. Regarding the histological analysis, the spleen tissue of the animals treated with SpHL-DOX or ExoSpHL-DOX was preserved A. The spTumor-cell-derived exosomes contain proteins and lipids similar to the cells that secrete them and fuse preferentially with their cells of origin, allowing for a higher concentration of drug delivery in the tumor . ExosomeIn conclusion, the results of the present study demonstrated that the toxicity of the ExoSpHL-DOX treatment is lower than the free DOX treatment, proving that exosome–liposome hybrid nanocarriers are capable of delivering higher doses of DOX without causing serious organ and tissue damage and with reduced adverse effects. In terms of the antitumor efficacy, the ExoSpHL-DOX treatment showed a higher antitumor effect compared to the control. Furthermore, ExoSpHL-DOX reduced the number of metastatic foci in the lungs, even when the exosome–liposome hybrid nanocarrier did not contain DOX. These results indicate that ExoSpHL-DOX may be a promising nanocarrier for the treatment of breast cancer, reducing toxicity and inhibiting metastasis, mainly in the lungs.Oliveira, M.C.; Gomes, E.R.; Sabino, A.P. “Composição farmacêutica contendo doxorrubicina encapsulada em vesículas híbridas de exossomas tumorais e lipossomas pH-sensíveis, processo e uso”. Instituto Nacional da Propriedade Industrial, nr. BR 10 2021 024659 6 (2021).


Antibody assays against SARS-CoV-2 are used in sero-epidemiological studies to estimate the proportion of a population with past infection. IgG antibodies against the spike protein (S-IgG) allow no distinction between infection and vaccination. We evaluated the role of anti-nucleocapsid-IgG (N-IgG) to identify individuals with infection more than one year past infection.S- and N-IgG were determined using the Euroimmun enzyme-linked immunosorbent assay (ELISA) in two groups: a randomly selected sample from the population of Stuttgart, Germany, and individuals with PCR-proven SARS-CoV-2 infection. Participants were five years or older. Demographics and comorbidities were registered from participants above 17 years.P = < 0.001). In vaccinated individuals, the mean signal ratios for S-IgG were higher in individuals with prior infection . N-IgG were only detectable in 17.1% of participants with past infection. Predictors for detectable N-IgG were older age, male sex, fever, wheezing and in-hospital treatment for COVID-19 and cardiovascular comorbidities.Between June 15, 2021 and July 14, 2021, 454 individuals from the random sample participated, as well as 217 individuals with past SARS-CoV-2 infection. Mean time from positive PCR test result to antibody testing was 458.7 days (standard deviation 14.6 days) in the past infection group. In unvaccinated individuals, the seroconversion rate for S-IgG was 25.5% in the random sample and 75% in the past infection group (N-IgG is not a reliable marker for SARS-CoV-2 infection after more than one year. In future, other diagnostic tests are needed to identify individuals with past natural infection.The online version contains supplementary material available at 10.1007/s15010-022-01830-x. Since its first emergence end of 2019, the new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally and caused several waves of infections around the globe . Since aAntibodies against viral structures that are not part as an antigen in the vaccines might help in this differentiation. For instance, in hepatitis B serological assays, hepatitis B core antibodies appear shortly after infection and persist for life . Since tThe nucleocapsid protein (NCP) of SARS-CoV-2 is vital for condensation and packaging of the viral genome and is the most abundant viral protein in infected cells , 6. NCP For the initial seroprevalence study, a representative random sample of 5000 inhabitants of the city of Stuttgart was drawn by the Statistisches Landesamt Baden-Württemberg  of inhabitants aged five years and older (= control group in this study). Besides the random sample, patients with PCR-proven SARS-CoV-2 infection were actively contacted by the local public health department. Letters containing an information sheet, declaration of consent and a questionnaire were sent to potential study participants or their legal representatives. There were three different versions of the information sheets and declaration of consents available for the age groups 5–12 years, 12–18 years, and 18 years and older. No questionnaire was sent to potential participants younger than 18 years. There was the possibility to complete the questionnaire online or to return a paper version back to the study site via mail or in person. All participants gave written informed consent before participation in the study. For individuals younger than 18 years, parents or a legal representative provided consent.For capillary blood sampling, the participants had the choice to either visit the study site or book an appointment where members of the study team visited them at home. There were three time points of data collection . In this study, only data from the third time point were used, since this was the only time point where N-IgG antibodies were analyzed. To study the seroconversion rate for N-IgG shortly after infection, sera from 20 patients that had COVID-19 during the second wave in November 2020 were analyzed. One patient was hospitalized and received supplemental oxygen. The others had mild to moderate symptoms.To study the humoral response after vaccination, an individual was considered fully vaccinated two weeks after they received the second shot of an mRNA vaccine  or the vector-based vaccine Vaxzevria (AstraZeneca). Participants receiving the COVID-19 vaccine Janssen (Johnson & Johnson) were considered fully vaccinated two weeks after the first shot, as only a single shot was recommended at the time of the study period. Participants with a heterologous Vaxzevria and mRNA prime-boost vaccination were also considered fully vaccinated two weeks after the second dose.After pricking of one of the fingertips with a lancet, blood was collected using the Multivette 600 blood collection system by Sarstedt . We assessed anti-SARS-CoV-2 nucleocapsid and spike IgG antibodies by enzyme-linked immunosorbent assays (ELISA) using a commercially available test kit . Samples were processed on a Dynex DS2 ELISA Processing System  according to manufacturer instructions. Results are reported semi-quantitatively by the ratio of extinction of the patient sample over the extinction of a calibrator. A signal was considered positive by calculation of a ratio of 1.1 or higher. The manufacturer considers a signal ratio of 0.8 to < 1.1 as borderline, which was considered negative in this study. A surrogate virus neutralization test (sVNT) was performed using a commercially available kit . Each assay was performed using an adequate positive and negative control, as recommended by the manufacturer. An inhibition rate of 30% or higher was considered positive for the detection of SARS-CoV-2 neutralizing antibodies. The assay has shown to have a specificity of 100% and a sensitivity of 98–98.6% using this cut-off  14.6 days) in the past infection group.From 15th of June 2021 to 14th of July 2021, 454 individuals from the randomly chosen sample from the population (= control group) participated, as well as 217 individuals with PCR-proven SARS-CoV-2 infection in the past. Patient characteristics and comorbidities are shown in (Table P < 0.01). On the contrary, significantly few people in the past infection group were smokers . Full vaccination rate was 67.3% in the control group and 77.3% in the past infection group. The difference was statistically significant (p < 0.05).Data on comorbidities were available for 302 participants (66.5%) in the control group and 162 (74.6%) in the past infection group, since questionnaires were only sent to individuals 18 years or older. More than half of the participants in both groups had no comorbidities (62.9% in the control group vs. 58.9% in the past infection group). Though, significantly more people in the past infection group had a respiratory comorbidity, such as chronic obstructive pulmonary disease or asthma . Signal ratios were significantly higher in the past infection group  compared to the control group  (p < 0.001)  with detectable S-IgG by ELISA , to determine the neutralizing activity of the antibodies. In general, there was a good positive correlation between the signal ratios as determined by S-IgG ELISA and inhibition rates in sVNT (p < 0.001) . Inhibition rates in sVNT were significantly higher in the past infection group  compared to the control group  (p < 0.001), indicating higher neutralizing antibody titers.In the control group, 71.6% of participants had detectable anti-SARS-CoV-2-Spike-IgG (S-IgG) and 94% in the past infection group (p < 0.001). In vaccinated individuals, the signal ratios for S-IgG were higher after prior infection , when compared to individuals without prior infection  .The seroconversion rate for S-IgG  was 94 and 99.4% for vaccinated individuals in the control and past-infection group, while it was 25.5 and 75% respectively in the unvaccinated group (p < 0.001). No difference was found in comparison to the heterologous vaccination group . Time from last vaccination to sample collection was not different between the groups . Signal ratios for S-IgG were significantly lower in participants receiving viral vector vaccines compared to mRNA and vaccines and after heterologous vaccination. No difference was seen between mRNA vaccines and heterologous vaccination  were vaccinated with Comirnaty (BioNTech-Pfizer), 64 16.5%) with Spikevax (Moderna), 74 (19.1%) with Vaxzevria (AstraZeneca), 3 (0.8%) with COVID-19 vaccine Janssen (Johnson & Johnson) and 14 (3.7%) had a heterologous vaccination . For analysis purposes, the mRNA vaccines Comirnaty and Spikevax were categorized as “mRNA vaccines” and the vaccines Vaxzevria and COVID-19 vaccine Janssen as “viral vector vaccines”. Patients in the viral vector vaccine group were significantly older  than patients in the mRNA vaccine group . Neutralizing antibody titers were also significantly higher after vaccination with mRNA vaccines . Because of only few participants with heterologous vaccinations, no sVNT was performed in this group.On a subgroup of patients, inhibition rates by sVNT, as an indicator for neutralizing antibody titers, were measured (p < 0.001). Mean signal ratios were significantly higher in the past infection group  compared to the control group .In the control group, 4.8% of individuals had detectable N-IgG and 17.1% in the past infection group (p = 0.25). Since N-IgG levels were not followed longitudinally, we wanted to rule out the fact that only a minority of individuals seroconverted shortly after infection, thereby explaining the low detection rate of N-IgG after more than one year. Therefore, N-IgG levels were determined in 20 individuals shortly after COVID-19 (range 14–47 days after positive PCR test result). Seroconversion rate for N-IgG was 100% with a mean signal ratio of 3.54 (range 1.18–7.08).Vaccination status had no influence on the seroprevalence of N-IgG antibodies  with PCR-proven SARS-CoV-2 infection more than one year ago had no detectable N-IgG antibodies. To answer the question what predicts N-IgG in the group of patients with known prior infections, we looked at different characteristics, as summarized in Table .9% with p < 0.001). Furthermore, few patients with N-IgG antibodies were female . Patients with fever above 38 °C  and with wheezing  during their SARS-CoV-2 infection had a higher probability of N-IgG positivity more than one year after infection. The same applies to participants with known cardiovascular comorbidities . Patients that were treated in-hospital due to COVID-19 had a higher seroprevalence rate for N-IgG antibodies .Individuals with detectable N-IgG antibodies were significantly older  in comparison to participants without detectable N-IgG  antibodies more than one year after infection. These antibodies are only detectable after prior infection, while anti-spike-IgG (S-IgG) are also elicited after vaccination.Here we report new insight into our understanding of the role of N-IgG in detection of individuals with prior SARS-CoV-2 infection. In this study, we show that N-IgG antibodies are only detectable in around 17% of patients around 14 months after PCR-proven infection. Predictors of N-IgG positivity are older age, male sex, cardiovascular comorbidities, fever and wheezing during infection, as well as in-hospital treatment for COVID-19. Furthermore, we show that vaccination leads to the induction of high levels S-IgG antibodies. A single dose after prior infection elicits higher S-IgG titers than two doses without prior infection. Also, mRNA vaccines and heterologous vaccination lead to higher S-IgG antibody titers in comparison to vector-based vaccines.Data on durability of N-IgG antibodies after COVID-19 are conflicting. The group by Shi et al. looked at the different dynamics of anti-SARS-CoV-2 antibodies over time . After oOn the other hand, Herrington et al. calculate an estimated time to sero-reversion for N-IgG antibodies of 18.6 days for 50% of individuals . Though,Older age is associated with a higher probability for N-IgG positivity more than one year after infection in our study. The mechanism is unknown, but Amjadi et al. show that older age is associated with higher anti-SARS-CoV-2 antibody levels, including N-IgG, after infection . AccordiOur study has several limitations. First, since N-IgG were only studied once and not longitudinally, we cannot make any conclusions about the trajectory of N-IgG antibodies after infection. Still the quintessence remains that N-IgG antibodies are not a reliable assay to determine past SARS-CoV-2 infection more than one year after infection. Second, another limitation is that in some subgroups, there were only a limited number of events  reducing the power of the study and increasing the margin of error. This can partially be explained by the fact that questionnaires were only answered by participants 18 years or older, leading to missing data by younger participants. Additionally, filling in the questionnaire was voluntary. The sole motivation for some of the participants was probably the determination of the antibody status. So, some participated in the blood sampling, without answering the questionnaire online or on paper. This is especially true for data about the vaccination status. Though, this does not impair our analysis since we mainly focused on the serological response after vaccination and are putting no spotlight on the level of immunity in the population. Third, in this study, we only use antibody assays from one company . Using N-IgG from different manufacturers, which themselves use other epitopes on the NCP or different antibody classes might lead to different results.A strength of our study is that it describes as one of few studies, the humoral immune response more than one year after SARS-CoV-2 infection. So far, most of the published studies looked at time frames of 12 months or below. We show that nucleocapsid antibodies become undetectable in most patients more than one year after infection. The quickly waning antibody response to NCP, especially in younger individuals with mild infection, would lead to underestimation of past SARS-CoV-2 infection. This has direct implications for the future design of sero-epidemiological studies, where relying on N-IgG antibodies would lead to a false estimation of individuals with prior SARS-CoV-2 infections in the population. Though, the identification of individuals with natural infections has some important implications since we also show that the humoral immune response after vaccination is more pronounced after prior infection. To identify such individuals, where probably vaccines with a lower antigen dose or less doses are sufficient, better assays to identify past natural infection are needed. B-cell epitopes located in the spike and nucleocapsid protein of the virus have shown to be the most reactive, with the lowest chances of cross-reactivity to other coronaviruses, suggesting that antibodies against these two proteins might still be preferred for future diagnostic tests for the humoral response against SARS-CoV-2 .In conclusion, we demonstrate that anti-nucleocapsid-IgG antibodies are not a reliable marker for prior SARS-CoV-2 infection after more than one year after infection. We show that the anti-spike-IgG antibody response is more durable but allows no distinction between prior infection and vaccination. In future, other diagnostic tests are needed to identify individuals with past natural infection after a longer time period.y-axis shows the inhibition rate in percent, measured by a surrogate viral neutralization assay. The dotted line represents the cut-off at 30% inhibition. Values 30% or above are considered positive. The x-axis shows the signal ratio for anti-spike-IgG antibodies measured by ELISA. (TIF 332 KB)Supplementary file1 Correlation between Spike-IgG signal ratios and inhibition rate measures by surrogate viral neutralization assay. 296 specimens are shown in this graph. A circle represents unvaccinated individuals, a cross individuals with mRNA vaccines and a triangle individuals with viral vector vaccines. The Below is the link to the electronic supplementary material.


Staphylococcus aureus implant-associated infections are difficult to treat because of the ability of bacteria to form biofilm on medical devices. Here, the efficacy of Sb-1 to control or prevent S. aureus colonization on medical foreign bodies was investigated in a Galleria mellonella larval infection model. For colonization control assays, sterile K-wires were implanted into larva prolegs. After 2 days, larvae were infected with methicillin-resistant S. aureus ATCC 43300 and incubated at 37 °C for a further 2 days, when treatments with either daptomycin (4 mg/kg), Sb-1 (107 PFUs) or a combination of them (3 x/day) were started. For biofilm prevention assays, larvae were pre-treated with either vancomycin (10 mg/kg) or Sb-1 (107 PFUs) before the S. aureus infection. In both experimental settings, K-wires were explanted for colony counting two days after treatment. In comparison to the untreated control, more than a 4 log10 CFU and 1 log10 CFU reduction was observed on K-wires recovered from larvae treated with the Sb-1/daptomycin combination and with their singular administration, respectively. Moreover, pre-infection treatment with Sb-1 was found to prevent K-wire colonization, similarly to vancomycin. Taken together, the obtained results demonstrated the strong potential of the Sb-1 antibiotic combinatory administration or the Sb-1 pretreatment to control or prevent S. aureus-associated implant infections. Staphylococcus aureus is one of the leading pathogens frequently involved in device colonization. S. aureus is responsible for 35.5% of prosthetic joint infections (PJIs) [S. aureus strains isolated in PJIs display biofilm-forming ability [S. aureus biofilm is refractory to antimicrobial compounds and the host immune system, making the eradication of implant-associated infections rather difficult [S. aureus (MSSA), the intravenous administration of oxacillin or cefazolin is the recommended treatment option [S. aureus (MRSA), the treatment includes vancomycin or daptomycin. Moreover, rifampin is added to the above-mentioned antibiotics to treat MRSA infections in patients undergoing debridement with retention or implant re-implantation in one- or two-stage exchange [S. aureus strains [S. aureus, including antibiotic-resistant isolates [Galleria mellonella larvae by preventing or treating MRSA infection compared to the untreated control [Indwelling medical devices, such as cardiac and orthopedic implants, are increasingly used in modern medicine to replace a compromised biological function or missing anatomical structure . Howevers (PJIs) . Moreoveifficult . Successexchange . As the  strains , a progr strains . Staphylisolates . In our  control . Moreove control . Notably control .G. mellonella larvae implanted with short stainless steel Kirschner-wires (K-wires). The phage–antibiotic combination determined a strong reduction in bacteria colonizing the K-wires in comparison to the single treatment administration. In addition, the in vivo ability of Sb-1 to prevent implant-associated infection due to MRSA in comparison to vancomycin was also investigated in the same G. mellonella model, showing comparable results between the prophylactic effect of phage and antibiotic.Here, we evaluated the in vivo efficacy of Sb-1 alone and in sequential combination with daptomycin against MRSA ATCC 43300 by using a foreign-body model of S. aureus is prone to colonize and form biofilm on medical implants, including stainless steel devices [4 to 108 PFU/mL) at 37 °C. After 24 h incubation, the number of MRSA ATCC 43300 CFUs that remained on the material was determined. Sb-1 treatment of biofilm samples resulted in a reduction of ≈2 log10 in the MRSA ATCC 43300 CFUs compared to the untreated control (no phage), even at the lowest titer tested (8 PFU/mL) was able to reduce the CFU number by more than 3 log10 compared to the untreated control, defined as the minimal biofilm bactericidal titer according to our previous works [ devices . To evalr tested . Howeverus works .S. aureus infection model was developed in G. mellonella larvae by using thin K-wire (cut in short pieces), which resulted in a suitable implant and was easily insertable into larvae. To assess the optimal MRSA ATCC 43300 infectious dose, allowing K-wire colonization two days after infection, K-wire implanted larvae were inoculated with different bacterial inocula, ranging from 104 CFU to 106 CFU. 4 CFU MRSA ATCC 43300, only three out of ten larvae tested showed K-wire colonization. Similarly, MRSA ATCC 43300 was found only on three out of ten K-wires implanted in larvae infected with 106 CFU bacteria, while the other seven larvae were dead within 24 h of infection. When larvae were injected with 105 CFU MRSA ATCC 43300, all larvae survived, and K-wires were colonized by bacteria with a median of 5.8 × 106 CFU/K-wire. Thus, 105 CFU was selected as the infectious dose for following in vivo experiments of infection treatment and prevention efficacy based on phages.The in vivo implant-associated 8 PFU/mL) were injected into larvae, five larvae were sacrificed at different time points and hemolymph was collected for phage counting by plaque assay. As shown in 8 PFU/mL Sb-1 was observed for up to 6 h, while after 24 h the titer of phages in larvae was drastically reduced (≈4 log10).Previous in vivo experiments performed in mice showed that the number of circulating active phages (able to lyse bacteria) reduced over time . A kinetS. aureus is known to easily colonize and form biofilm on medical indwelling devices, resulting in an infection that is difficult to treat with current antibacterial therapies. In order to evaluate the ability of Sb-1  to reduce the in vivo bacterial colonization of K-wires, larvae were infected with 105 CFU MRSA ATCC 43300 and treated three times/day for 2 days with 107 PFUs Sb-1 and/or 4 mg/kg daptomycin. In these assays, daptomycin was selected for combinatory treatment, as it is the antibiotic currently used in combination with rifampin for the management of MRSA implant-associated infection [10 CFUs of medians was observed when larvae were treated with Sb-1 alone for either 48 or 24 h. Similar results were obtained when larvae were treated with daptomycin, suggesting that Sb-1 had a lytic activity similar to that exhibited by the antibiotic. A reduction of more than 3 log10 CFU/K-wire number was observed when larvae were treated with a staggered combination of phages and antibiotic in comparison to phages or antibiotic administered singularly. Moreover, a stronger reduction in CFU/K-wire number ≈ 4 log10 was obtained in comparison to the untreated control.nfection . Figure 7 Sb-1 in preventing K-wire colonization by MRSA ATCC 43300 was also tested and compared to the efficacy of vancomycin (10 mg/kg), the antibiotic used for the prevention of surgical site infection (10). Prophylactic treatment with either phages or antibiotics was administered as a single dose in implanted larvae 1 h earlier than MRSA ATCC 43300 infection. At two and five days post-infection, larvae were sacrificed and for all of them the K-wire was removed for the MRSA CFU count. In parallel, the hemolymph was collected to evaluate the number of circulating bacteria. As an established biofilm is difficult to eradicate, the prevention of the infection is still considered the best strategy to reduce the cases of infection . TherefoS. aureus, to colonize materials and to form biofilm on implanted medical devices represents a further challenge for the treatment of chronic staphylococcal infections, as only a few antibiotics administered in combination are active against sessile bacteria [G. mellonella model of larvae implanted with stainless steel K-wires. In recent years, G. mellonella larvae have been proposed as a suitable in vivo model for investigating the virulence of different microorganisms, including human pathogens, and for evaluating the toxicity and efficacy of antimicrobial compounds [The increase in antibiotic resistance and the lack in the pipeline of antimicrobial molecules with novel mechanisms of action make bacterial infections increasingly difficult to treat . Moreovebacteria , especiabacteria . In thisompounds . Mostly,ompounds ,20.in vitro. After 24 h incubation, 108 PFU/mL Sb-1 resulted in a bactericidal effect against the staphylococcal biofilm. These results were consistent with data that we observed in previous studies where Sb-1 showed a characteristic antibiofilm activity, strongly reducing CFU/mL numbers of sessile bacteria, degrading the matrix and targeting metabolically active and persister cells of MRSA ATCC 43300 biofilm formed on porous glass beads [First, we observed the ability of MRSA ATCC 43300 to colonize K-wires in vitro and we confirmed the lytic ability of Sb-1 versus sessile cells ss beads .G. mellonella larvae for the following in vivo experiments. An inoculum of 105 CFUs allowed the in vivo K-wire colonization and larval survival. By contrast, a higher tested inoculum of MRSA (106 CFUs) was shown to be fatal for larvae at 24 h post-infection, as previously reported by the authors [G. mellonella model of implant-associated infection caused by MRSA ATCC 43300, which might be used not only to test novel compounds, but also to investigate and provide new insights into the dynamic of the in vivo colonization and biofilm formation.Due to their use in surgery, together with their small dimension and the ability of MRSA ATCC 43300 to colonize and grow on it, stainless steel K-wires were considered a good implant candidate to be used in  authors ,22. ThusP. aeruginosa biofilm [S. aureus ATCC 43300 attached to a material was also observed in G. mellonella larvae implanted with K-wires. A group of larvae was treated with daptomycin, which was used as the control of standard treatment, as it is considered a first-choice drug in the clinical practice for methicillin-resistant S. aureus biofilm infection [A critical point for the in vivo treatment of infected K-wire was the phage titer/doses and the number of doses/days to be administered to infected larvae. As we had no previous information regarding phage pharmacokinetics in larvae, the number of phages circulating in the hemolymph of uninfected larvae at different time points after injection was evaluated. The kinetic of clearance and/or inactivation of Sb-1 in larvae showed that the number of lytic phages decreased at 6–12 h post inoculum. Therefore, we decided to administer phages three times/day in order to keep a constant phage titer circulating in the haemolymph. Three times/day was also the dose reported for the phage treatment of patients suffering from prosthetic joint infections due to  biofilm . In agrenfection ,6. A simS. aureus sessile cells, even at a lower concentration of antibiotic, we verified that the same efficacy also occurred in vivo. The strong reduction in viable S. aureus cells observed in this study in the group of larvae sequentially treated with Sb-1 and daptomycin confirmed the bactericidal activity of the Sb-1- daptomycin combinatory treatment previously observed in vitro [S. aureus is known to rapidly develop resistance to rifampin [S. aureus biofilm, and in combination with daptomycin, as an alternative to the RNA Polymerase inhibitor drug, for the treatment of MRSA infection.Based on our previously published evidence  showing in vitro , and it in vitro ,24, but in vitro  suggestsrifampin . For thiAntimicrobial prophylaxis is currently recommended during orthopedic surgery in which a device is implanted. This consists in the administration of first- or second-generation cephalosporin in all patients, and a supplemental vancomycin administration in MRSA carriers .S. aureus by Sb-1 decreases the initial bacterial load, and consequently it reduced the chance of bacteria to colonize the implanted K-wire and establish an infection in G. mellonella larvae. Similar results were also obtained for vancomycin, suggesting that Sb-1 might also be used in human therapy as a prophylaxis of surgical site infection instead of antibiotics. In general, phages have a rather narrow host range even at strain level; however, Sb-1 has been shown to be active against ≈90% of diverse global S. aureus isolates [S. aureus.The results obtained suggested that the fast lysis of methicillin-resistant S. aureus cells colonizing stainless steel implants has been demonstrated in vivo. This evidence might spur the design and roll-out of clinical trials to validate the efficacy of phage-based treatments as an adjuvant of antibiotic therapy for the eradication of implant-associated infection due to drug-resistant S. aureus.In conclusion, the potential activity of Sb-1 treatment in combination with antibiotics against S. aureus ATCC 43300 was used in this study. Bacteria were stored in a cryovial bead preservation system  at −80 °C and cultivated on Brain-Heart Infusion  agar plates. Brain-Hearth Infusion broth was used for bacterial inoculum and phage production. Biofilm sonication fluids from larval infection experiments were plated on mannitol salt agar .The methicillin-resistant laboratory strain S. aureus ATCC 43300 was used as host to amplify Sb-1. Briefly, an overnight culture of S. aureus ATCC 43300 was diluted 1:100 in fresh medium with 0.1 M MgSO4  and incubated at 37 °C until it reached 0.2–0.3 optical density (OD) corresponding to ≈ 1 × 107 CFU/mL for S. aureus. Then, bacterial culture was infected with Sb-1 at a multiplicity of infection (MOI) of 1:10. ODs were monitored at different time points until the values reached the OD values obtained before phage infection. Bacterial cultures were centrifuged (4000× g for 15 min at 4 °C) and the supernatant was filtered through a 0.22 µm syringe filter . The phage titer was determined by plaque assay.A commercially available formulation of Staphylococcal bacteriophage Sb-1  was obtained as a 10 mL liquid solution. 2HPO4 and 2 mM KH2HPO4, pH 7.5) by 10-fold serial dilutions in order to count plaque numbers. Briefly, 0.5 mL bacteriophage lysate of each dilution and 0.3 mL host bacteria from an overnight culture were added to 3 mL top agar  at 50 °C and immediately poured onto Brain-Heart Infusion agar plates. Two plates for each dilution were used. After overnight incubation of plates at 37 °C, plaques were counted and the titer was calculated as plaque-forming unit (PFU)/mL. Phages were stored at 4 °C.Phage lysate was diluted in phosphate-buffered saline  glucose . After 24 h incubation, K-wires were rinsed 3 times using sterile PBS to remove planktonic bacteria. For CFU counting, biofilm-embedded bacteria were dislodged from K-wires by placing each of them in a 2 mL Eppendorf tube with 1 mL PBS and treating them by sequential steps of vortexing (30″), sonicating (1′) and vortexing (30″), as previously reported [8 PFU/mL to 104 PFU/mL). S. aureus biofilm incubated with an equal volume of PBS served as control. After incubation with Sb-1, K-wires were rinsed 3 times with PBS to remove phages and unattached bacteria. For colony counting, samples were sonicated and plated as previously described.Stainless steel Kirschner-wires (K-wires)  (0.6 mm diameter) were cut into 4 mm length pieces and sterilized by autoclaving. Then, reported . The obtG. mellonella bred without antimicrobials or hormones were obtained from BioSystems Technology Ltd. . On their arrival, larvae were stored at room temperature in the dark to prevent them from turning into pupae and used within 3 days. Stainless steel K-wires , thin pins used in orthopedic surgery to stabilize bone fragments, were implanted in the last left proleg of larvae by using an insulin syringe needle to create a hole for the entrance of the pins. Then, larvae were left to set for 2 days at RT before infection  in the last right proleg, on the opposite side where a k-wire was inserted. Two days post-infection, larvae were sacrificed to collect the K-wires, which were sonicated as previously described. Then, sonication fluids were plated for colony counting as reported above. To evaluate the stability of circulating Sb-1 in G. mellonella, larvae were injected into the last left proleg with 108 PFU/mL phages and, after that, 5 larvae per group were sacrificed at different time points  to collect the hemolymph and to count phage titer by plaque assay  for 48 h, (ii) daptomycin (4 mg/kg) for 48 h, (iii) 107 PFU Sb-1 for 24 h followed by 4 mg/kg daptomycin for further 24 h, (iv) Sb-1 (107 PFUs) for 24 h followed by PBS for further 24 h, and (v) PBS for 24 h followed by Daptomycin (4 mg/kg) for further 24 h. An untreated group of S. aureus-infected larvae was included as positive control of biofilm formation. Phage and daptomycin doses were chosen according to their use in the clinical practice [In order to evaluate the ability of phage to treat MRSA biofilm-associated infection, larvae implanted with K-wire and infected with MRSA ATCC 43300, as previously described, were treated with 10practice ,28 and pG. mellonella larvae (10 for each group) were infected with 105 CFUs MRSA ATCC 43300 on the same day of K-wire implant and 1 h after either Sb-1 (MOI 100) or vancomycin (10 mg/kg) administration according to the procedure showed in To evaluate the efficacy of prophylactic application of Sb-1 to prevent MRSA colonization on K-wires, G. mellonella larvae was performed by using the non-parametric Kruskal–Wallis test using Dunn’s correction. For all tests, differences were considered significant when p values were <0.05. Graphs were plotted and analyzed with Prism software .Statistical analysis of Sb-1 treatment and prevention data obtained from implanted